A novel assay has been developed for studying this site-specific recombination in vitro. The substrate (developed by H. Nash) is a lambda DNA containing two sets of attachment sites; recombination excises the fragment between the sites. The assay looks at the sizes of restriction fragments produced by the Eco R1 enzyme. The recombined products have different fragment sizes, as measured by electrophoresis in agarose gels, from the starting DNA. Using this assay, the following results have been obtained: 1) Covalently circular DNA is the only effective substrate. Linear DNA is inactive; nicked circular DNA works only if it can be sealed by DNA ligase in the reaction mixture. 2) Both recombination products (the excised fragment and the remaining lambda DNA) are recovered in equal yield. 3) Both recombination products are recovered largely in the form of covalent circles, even when DNA ligase in the cell extracts is inhibited by NMN. This raises the possibility of a ligase-independent sealing step as a coupled part of the recombination process. 4) If the products are not treated with restriction enzyme, most of the excised fragment is recovered as circles interlocked (catenated) with the circular lambda DNA product. This result implies that recombination occurs while the substrate DNA is supercoiled.